1. Field of the Invention
This invention relates to reagent compositions useful in the flow cytometric analysis of blood cells, particularly related to a lytic reagent composition enabling differentiation of leukocyte into five subpopulations.
2. Prior Art
Examination and analysis of the cellular types and subtypes of human and animal whole blood specimens is an important part of modern healthcare. For example, higher or lower numbers of red blood cells (erythrocytes), white blood cells (leukocytes) and platelets are consistent with diagnosis of certain disease states, as well as useful indicators of recovery from the diseases. Traditionally, these tests have been performed by direct counting and size measurement of the cellular constituents by means of a volume-calibrated glass microscope chamber, or more recently by means of electronic counting of the cellular constituents suspended in a supportive, electrically conductive solution. Determination of white blood cell number has been performed in a similar manner, but direct electronic determination of white blood cell subpopulations has only relatively recently become possible by means of electronic size discrimination analysis.
This latter approach to leukocyte differential analysis has been further improved by use of flow cytometric analysis. In this procedure, blood cells are suspended in a reagent solution and passed through a flow cell and detected by an electro-optical detector. The cells are then classified on the basis of one or more measurable parameters, including cellular volume determination by an impedance method, cellular granularity by an RF conductivity method, light scattering or light absorption. The advantages of this type of flow cytometric system over direct microscopic examination is a much higher throughput of specimen samples and the much larger numbers of cells analyzed per sample, thus improving the counting and sizing statistics and reducing the contribution of non-specific noise.
Analyzers using flow cytometric analysis differentiate and classify leukocyte subpopulations by use of light scatter or by electrical impedance parameters, or both. For proper function and sensitivity of the leukocyte analysis, the more numerous red blood cells in the whole blood specimen are usually removed by chemical lysis in order to reveal the much smaller number of white blood cells. For electrical impedance-based analyzers, stronger lysing reagents, such as quaternary ammonium surfactant based reagents, have been used for differentiating leukocytes into two or three subpopulations by sizing. However, such lysing reagents cause serious leukocyte membrane damage, rendering the differential analysis unsuitable when the blood sample is analyzed by flow cytometric systems employing light scattering and light absorbance measurements. For these types of measurement systems, lytic reagents should provide rapid red cell lysis, but not unduly disturb the light scattering or absorption characteristics of leukocytes for a time long enough to collect optical data for the differential analysis.
It is known that blood cell lysis proceeds more rapidly at alkaline pH than at neutral or acidic pH. Lytic reagents with very alkaline or very acidic pH typically can lyse red blood cells, as well as white blood cells, rendering the leukocyte differential analysis difficult. Reagents with near neutral pH are too slow, requiring several minutes to complete the lysing reaction.
U.S. Pat. No. 4,617,275 (to Matsuda, et al.) describes the use of a lysing reagent comprising quaternary ammonium salts to provide adequate red cell lysis without excessively damaging the white blood cells for the purpose of electrical impedance measurement of at least three subpopulations of leukocytes. The lysing reagent contains citric acid to assist in removal of the interfering red cell ghosts. The analysis method requires the use of a diluent solution as a co-reagent. The diluent contains a buffer comprised of boric acid and sodium borate.
U.S. Pat. No. 4,637,986 (to Brown, et al.) describes a flow cytometry lysing reagent for producing a 3-part differential of leukocytes. The lysing reagent is a hypotonic aqueous solution enabling hypotonic lysis of red blood cells. The lysing reagent comprises a leukoprotective agent for preserving the lymphocyte cellular integrity during analysis, and buffers to provide the correct pH environment for optimal lysis. The preferred leukoprotective agents include methyl oxyethanol, ethyl oxyethanol and butyl oxyethanol. Brown et al. specifically teach the importance of the pH range of the lysing reagent in order to provide rapid lysing of red blood cells, but prevent deterioration of the leukocytes. More specifically, the preferred pH range is from approximately 8.1 to about 8.8, with optimal around 8.5. Brown et al. teach that if pH of the lysing reagent is above 9.0, a rapid deterioration of the white blood cells occurs, which results in disadvantageous effects on counting accuracy. Furthermore, Brown et al. teach that the specific buffering agent is critical to the proper operation of the lysing agent. More specifically, it is found that mineral buffers such as phosphate and carboxylic acids or molecules containing a carboxyl group are not effective, and the boric acid/TRIS buffer combination is also less effective. Brown et al. specifically teach the optimal buffer system of an organic, nitrogen containing buffer balanced with an ethane sulfonic acid buffer to maintain pH of the lysing reagent from 8.1 to 8.8.
U.S. Pat. No. 5,510,267 (to Marshall) describes a flow cytometry lytic reagent and a method for providing a 5-part differential analysis of leukocytes. The method includes diluting a blood sample with a neutral and near isotonic diluent, mixing the diluted sample with the lytic reagent to lyse red blood cells, and analyzing the sample mixture in a flow cell by measuring 0°, 10°, 90° and 90° depolarized light scatter signals to differentiate leukocytes into five subpopulations, including neutrophils, lymphocytes, monocytes, eosinophils and basophils. Marshall teaches that the lytic reagent includes 2-phenoxyethanol which combines the function of leukoprotective and antimicrobial; Triton X-100 (octylphenoxypolyethoxyethanol) a lytic and wetting agent; and an organic buffer with pKa at or near 8.5 to maintain pH of the lysing reagent at 8.5. Furthermore, Marshall teaches the importance of pH to the function of the lytic reagent. More specifically, the optimal pH is 8.5, and with a lower range of 8.1 without significant effects on the reagent performance. However, if pH of the lytic reagent increases to 9.0, partial destruction of white blood cells can occur.
Both U.S. Pat. No. 4,637,986 and U.S. Pat. No. 5,510,267 teach that red blood cell lysis under alkaline condition is more rapid, which is desirable for automated hematology analyzers. However, at pH above 9, white blood cell damage occurs, which impacts the accuracy of the leukocyte counting and differential analysis.
Therefore, it is apparent that there is a need for an improved lytic reagent composition which enables rapid red blood cell lysis under alkaline condition, but enables preserving the leukocytes for leukocyte differential analysis using automated hematology analyzers.